Parathyroid hormone (PTH) modulates the activity of bone and kidney cells via interaction with specific receptors (PTH-R) located on the external membrane surface of target cells. PTH binding triggers activation of G proteins that regulate activity of several signal generating systems, including adenylyl cyclase, phospholipase C, and intracellular calcium. The human disease pseudohypoparathyroidism type I (PHP type I) provides a model for the physiologic relevance of PTH-mediated signal transduction. PHP type I is characterized by biochemical hypoparathyroidism (i.e. hypocalcemia and hyperphosphatemia) due to target tissue resistance to the biological actions of PTH. Administration of PTH to patients with PHP type I does not increase urinary excretion of nephrogenous cyclic AMP, suggesting that PTH resistance is caused by a defect in the PTH receptor-adenylyl cyclase complex. In PHP type Ia a generalized deficiency of the alpha subunit of the G protein (G(s)alpha that couples stimulatory receptors to activation of adenylyl cyclase is associated with not only PTH resistance, but with widespread resistance to multiple hormones that act via stimulation of adenylyl cyclase. By contrast, a defect in the PTH-R would explain PTH resistance in other patients, termed PHP type Ib, who have normal levels of G(s)alpha protein and show hormone resistance that is limited to PTH target tissues. Our goal over the next 5 years is to identify and characterize defects in the PTH-R gene that lead to decreased PTH-R function in patients with PHP type Ib. We will clone cDNAs encoding human PTH-R(s) and use these sequences to analyze genomic DNA and mRNA from subjects with PHP type Ib. We will perform linkage analysis to determine whether inheritance of specific PTH-R gene alleles is associated with PHP type Ib. Restriction enzyme analysis of genomic DNA will be used to identify deletions, insertions, or rearrangements within the PTH-R gene. To identify small (e.g. point) mutations we will use denaturing gradient gel electrophoresis to analyze regions of the PTH receptor mRNA or gene that have been amplified by the polymerase chain reaction. To determine the functional consequences of changes in the primary structure of the PTH-R, mutant PTH-R proteins will be expressed in COS cells, which genetically lack endogenous PTH-Rs, and the ability of the mutant receptor to bind PTH and activate adenylyl cyclase, phospholipase C, and calcium signals will be assessed. The molecular delineation of mutations in the structural of the PTH-R in patients with PHP type Ib will ultimately provide insights into the function of specific structural domains of the PTH-R protein. We predict that characterization of defects within the PTH-R in patients with PHP type Ib will further our understanding of regions within G protein-linked receptors that determine specificity of interaction between receptors and G proteins.